Pan-cancer Gene Panel Research Articles

SJPedPanel: A Pan-Cancer Gene Panel for Childhood Malignancies to Enhance Cancer Monitoring and Early Detection.

The purpose of the study was to design a pan-cancer gene panel for childhood malignancies and validate it using clinically characterized patient samples. In addition to 5,275 coding exons, SJPedPanel also covers 297 introns for fusions/structural variations and 7,590 polymorphic sites for copy-number alterations. Capture uniformity and limit of detection are determined by targeted sequencing of cell lines using dilution experiment. We validate its coverage by in silico analysis of an established real-time clinical genomics (RTCG) cohort of 253 patients. We further validate its performance by targeted resequencing of 113 patient samples from the RTCG cohort. We demonstrate its power in analyzing low tumor burden specimens using morphologic remission and monitoring samples. Among the 485 pathogenic variants reported in RTCG cohort, SJPedPanel covered 86% of variants, including 82% of 90 rearrangements responsible for fusion oncoproteins. In our targeted resequencing cohort, 91% of 389 pathogenic variants are detected. The gene panel enabled us to detect ∼95% of variants at allele fraction (AF) 0.5%, whereas the detection rate is ∼80% at AF 0.2%. The panel detected low-frequency driver alterations from morphologic leukemia remission samples and relapse-enriched alterations from monitoring samples, demonstrating its power for cancer monitoring and early detection. SJPedPanel enables the cost-effective detection of clinically relevant genetic alterations including rearrangements responsible for subtype-defining fusions by targeted sequencing of ∼0.15% of human genome for childhood malignancies. It will enhance the analysis of specimens with low tumor burdens for cancer monitoring and early detection.

Mutational signatures of colorectal cancers according to distinct computational workflows.

Tumor mutational signatures have gained prominence in cancer research, yet the lack of standardized methods hinders reproducibility and robustness. Leveraging colorectal cancer (CRC) as a model, we explored the influence of computational parameters on mutational signature analyses across 230 CRC cell lines and 152 CRC patients. Results were validated in three independent datasets: 483 endometrial cancer patients stratified by mismatch repair (MMR) status, 35 lung cancer patients by smoking status and 12 patient-derived organoids (PDOs) annotated for colibactin exposure. Assessing various bioinformatic tools, reference datasets and input data sizes including whole genome sequencing, whole exome sequencing and a pan-cancer gene panel, we demonstrated significant variability in the results. We report that the use of distinct algorithms and references led to statistically different results, highlighting how arbitrary choices may induce variability in the mutational signature contributions. Furthermore, we found a differential contribution of mutational signatures between coding and intergenic regions and defined the minimum number of somatic variants required for reliable mutational signature assignment. To facilitate the identification of the most suitable workflows, we developed Comparative Mutational Signature analysis on Coding and Extragenic Regions (CoMSCER), a bioinformatic tool which allows researchers to easily perform comparative mutational signature analysis by coupling the results from several tools and public reference datasets and to assess mutational signature contributions in coding and non-coding genomic regions. In conclusion, our study provides a comparative framework to elucidate the impact of distinct computational workflows on mutational signatures.

Abstract PO5-13-05: Comparison of whole exome, whole transcriptome genomic profiling and targeted sequencing with 50-gene panels

Abstract Introduction Genomic profiling to identify targetable genomic alterations is the standard of care in the management of advanced cancers, including breast cancer (BC), and is recommended by ASCO provisional clinical guidelines. Genomic panels of ≤50 genes are generally less expensive than larger panels but may miss targetable alterations. This study compared a comprehensive tumor-normal whole-exome, whole-transcriptome panel to four 50-gene panels. In addition, for triple-negative BC (TNBC), the budget impact of using a comprehensive panel versus 50-gene panels was estimated. Methods Gene lists derived from three commercially available 50-gene panels (A, B and C) and a customized pan-cancer gene panel list (D) were used. The genes listed on the custom panel were chosen by medical experts. Samples from BC patients that underwent OncoExTraTM testing from May 2018 through March 2023 were included. The OncoExTra assay uses tumor-normal whole exome and whole transcriptome sequencing to detect somatic single base substitutions, indels, copy number alterations, gene fusions and alternative transcripts. In this analysis, the focus was actionable biomarkers, defined as those with associated FDA-approved targeted therapies in any cancer type, those that made patients eligible for an active clinical trial, or those with evidence in guidelines or the literature for possible matched therapies in any cancer. The frequencies of patients who would have been missed (no actionable alterations identified) using only the genes on each 50-gene panel were summarized. It was assumed that any alteration involving a listed gene would be detected, maximizing the potential utility of each panel. For TNBC, the budget impact of shifting the 20% of patients currently tested with 50-gene panels to OncoExTra testing was estimated using a previously published model. Results A total of 2161 BC patient samples were included. Across all samples, between 11.9% and 28.1% of patients would have been missed by restricting to only 50 genes (Table 1). The proportion of missed patients across panels was highest in TNBC and lowest in HER2+ cancers. Major drivers of missed patients included absence of FGF3/4/19 (A-D), MAP3K1 (A-D), BRCA1/2 (A, B, C), CCND1 (B, C, D), and PTEN (A, D). Across the four panels, patients were missed due to actionable alterations in 96 to 126 genes. Tumor mutational burden and microsatellite instability, which are not reported in 50-gene panels, were high in 35 (1.6%) and 7 (0.3%) BC patient samples, respectively. Within a 1-million-member plan, 44 TNBC patients would be eligible for testing. If all these patients were tested with OncoExtra rather than a 50-gene panel, the increase in per-member-per-month costs ranges from $0.0104-0.0172, driven predominantly by increased matched therapy costs. Between two and five TNBC patients would be eligible to receive a different treatment with a comprehensive panel. Conclusions In BC, between 11.9% and 28.1% of patients with actionable alterations would have been missed by restricting analysis to the 50 genes found in these panels. Using a comprehensive profiling assay such OncoExTra led to the identification of additional TNBC patients with actionable alterations at a minimal budget impact. Table 1. BC patients (pts) with actionable alterations missed by each 50-gene panel. NS = subtype not specified. Citation Format: Snehal Thakkar, David Hall, Jess Hoag, Cynthia Flannery, Nishitha Therala, Anson Tharayanil, Jesse Ortendahl, Gebra Cuyun Carter, Gargi Basu. Comparison of whole exome, whole transcriptome genomic profiling and targeted sequencing with 50-gene panels [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-05.

Abstract 2406: Circulating tumor DNA as a potential indicator of treatment response in patients with surgically unresectable PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly heterogeneous and extremely aggressive neoplasm. Most patients with PDAC present with symptomatic, surgically unresectable disease at initial diagnosis. Systemic chemotherapy is the standard care for patients diagnosed with unresectable PDAC, despite the dismal survival rate due to disease recurrence. In this context, liquid biopsy based ctDNA monitoring may aid to improve detection accuracy and allow real time disease prognosis. Here we aimed to investigate the utility of ctDNA in longitudinal tumor monitoring to assess treatment response and to predict disease recurrence. We performed targeted NGS sequencing to investigate over 900 mutations (SNVs) and 12 copy number variations (CNVs) in 53 genes, using the Ion S5 Oncomine PanCancer gene panel in 40 PDAC patients with unresectable disease. Serial plasma samples were obtained from patients in the course of 24 months, including samples collected before the first intervention (baseline) as well as within 2 months after initiation of chemotherapy. Patients were followed for a period of up to 2-years from diagnosis through treatment, to death or final follow-up. Genomic DNA from paired plasma-buffy coat samples were sequenced to exclude clonal hematopoiesis. Overall detection of ctDNA alterations in baseline samples was 55% (22/40) with increasingly higher detection rates and mutation loads in advanced disease (88% in stage IV patients). Alterations including SNVs and CNVs were preferentially distributed among 9 genes; mutations in TP53 (45%), KRAS (38%) and SMAD4 (5%) were most commonly identified. To asses response to chemotherapy, the presence of residual ctDNA after clinical intervention was evaluated by comparing total ctDNA MAF (mutant allele frequency) detected in plasma at diagnosis and within 2 months of chemotherapy. 71% (17/24) of patients who reached progressive disease (PD) had detectable baseline mutations and 79% (19/24) had persistent residual mutations at 2 months of treatment. Conversely, 94% (15/16) of non-PD patients had no detectable ctDNA or had a significant reduction in total MAF after 2 months of chemotherapy. Absence of residual ctDNA after 2 months of chemotherapy was associated with a better OS (p=0.031; HR = 3.48). On the other hand, residual ctDNA after 2 months of chemotherapy was associated with a shorter PFS (p=0.042; HR = 3.28). Our study demonstrates that monitoring ctDNA levels derived from plasma provides valuable insights into the therapeutic response in patients with unresectable PDAC, even in the absence of matched tumor genotyping. We found a significant correlation between increased ctDNA levels following the initiation of treatment and poorer survival outcomes. Citation Format: Fabio Pittella Silva, Yasutoshi Kimura, Masayo Motoya, Toru Nakamura, Takeshi Murakami, Kazunori Watanabe, Siew-Kee Low, Yusuke Nakamura. Circulating tumor DNA as a potential indicator of treatment response in patients with surgically unresectable PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2406.

Abstract 3898: SJPedPanel: A pan-cancer gene panel for childhood malignancies

Abstract Background: Extensive efforts in the past decade have revolutionized our understanding of the genetic underpinnings of childhood malignancies and identified numerous driver alterations that can provide potential targets for novel therapy and are excellent biomarkers for disease monitoring. For these purposes, a whole genome or exome sequencing approach can be resource prohibitive. Numerous gene panels are developed for adult cancers to address these challenges. Due to the dramatic differences in driver gene landscapes between pediatric and adult cancers, a gene panel for childhood cancers is needed. Results: Here, we have developed a gene panel dedicated to childhood cancers. This panel (2.8 Mbp) covers 5275 coding exons of 357 driver genes, 297 introns frequently involved in rearrangements that generate fusion oncoproteins, commonly deleted regions, such as CDKN2A and PAX5 (for B-/T-ALL) and SMARCB1 (for ATRT), and 7,590 polymorphism sites to detect copy number alterations for interrogating tumors with aneuploidy, such as hyperdiploid and hypodiploid B-ALL or 17q gain neuroblastoma. We used driver alterations reported from an established real-time clinical genomics cohort (n=253) to investigate the effectiveness of this gene panel. Among the 485 pathogenic variants reported, our panel covered 417 variants (86%). For 90 rearrangements responsible for oncogenic fusions, our panel covered 74 events (82%). We re-sequenced 113 previously characterized clinical specimens at an average depth of 2,500X using SJPedPanel and recovered 355 (90%) of the 396 reported pathogenic variants. Among the 30 unique genes of the 41 missed alterations, 29 genes are mutated in pediatric cancers with a low frequency (<0.21%) and hence not covered in the panel. We then investigated the power of this panel in detecting mutations from specimens with low tumor content (as low as 0.1%) using cell line-based dilution experiments and discovered that this gene panel enabled us to detect ~80% variants with allele fraction of 0.2%, while the detection rate decreases to ~50% when the allele fraction is 0.1%. We finally demonstrate its utility in disease monitoring on clinical specimens collected from AML patients in morphologic remission. Conclusion: Overall, our gene panel enables the detection of clinically relevant genetic alterations including rearrangements responsible for subtype-defining fusions for childhood cancers by targeted sequencing of ~0.15% of human genome. Our panel will significantly enhance the analysis of specimens with low tumor burdens for cancer monitoring and early detection. Citation Format: Pandurang Kolekar, Vidya Balagopal, Li Dong, Yanling Liu, Scott Foy, Quang Tran, Heather Mulder, Anna LW Huskey, Emily Plyler, Zhikai Liang, Jingqun Ma, Joy Nakitandwe, Jiali Gu, Jamie Maciaszek, Debbie Payne-Turner, Saradhi Mallampati, Lu Wang, John Easton, Jeffery M. Klco, Xiaotu Ma. SJPedPanel: A pan-cancer gene panel for childhood malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3898.

P10.24.B SCREENING NON-ONCOLOGICAL DRUGS IN A PATIENT-DERIVED GLIOBLASTOMA MODEL IDENTIFIES PITAVASTATIN WITH POTENTIAL ANTITUMOR ACTIVITY

Abstract BACKGROUND Glioblastoma (GBM) is a malignancy which is in dire need of novel treatment options. Repurposing non-oncological drugs has become of great interest in GBM research. Non-oncological drugs have many advantages over their oncological counterparts including a well-established safety and toxicity profile, possibilities to perform dose escalation studies, and combinations with oncological drugs (e.g. temozolomide, TMZ). Making use of our patient-derived in vitro model which has been shown to recapitulate GBM in terms of genetic make-up and heterogeneity, we aim at identifying non-oncological drugs that exhibit potent antitumor activity. MATERIAL AND METHODS Drug screening was performed using an NIH clinical compound selection consisting of 446 non-oncological drugs on 2 patient-derived GBM cell lines. Viability 5 days post treatment was assessed using the ATP-based Cell Titer Glo assay. Validation studies were performed on a panel of 25 GBM cultures. Nanostring analysis using the Pan Cancer gene panel, flow cytometry, Western Blotting and immunofluorescence were employed to gain insight into drug mechanism of action. The zero-interaction potential method was used to quantify synergy with TMZ. Dose-modifying factors (DMF) were calculated to quantify synergy with irradiation. RESULTS Screening of 446 non-oncological drugs yielded a top 10 list of candidates which were evaluated on an additional cell panel. Based on safety and efficacy, pitavastatin, an HMG-CoA reductase inhibitor, was selected for further analysis. Compared to 8 other registered statins, pitavastatin revealed the most potent anti-tumor activity, with IC50 values in the low micromolar range. Pitavastatin efficacy was validated in a panel of 25 GBM cultures derived from various GBM subtypes in terms of primary/recurrence, MGMT and IDH status (IC50 ranging from 0.1 - 5.0 µM). GBM cultures derived from patients using statins for cholesterol management did not reveal resistance to pitavastatin. Combination treatment of pitavastatin with either TMZ or irradiation showed synergy with TMZ in 5/8 cultures and radiosensitizing effects in 3/5 cultures (DMF >2.0). Importantly, no antagonism was observed with TMZ or irradiation. Mechanistic studies demonstrated that pitavastatin treatment led to induction of autophagy, perturbations in GBM cell cycle and attenuation of GSC stemness through downregulation of SOX2 and CD133. CONCLUSION Drug screening on patient derived GBM cultures identified pitavastatin as a potent inhibitor of GBM cell viability and revealed its chemo- and radiosensitizing effects. Mechanistic studies suggest that its antitumor effects could be a consequence of autophagy induction, cell cycle arrest and loss of GBM cell stemness. Currently we are exploring strategies to ensure optimal intratumoral delivery in vivo.

Circulating Tumor DNA: A Promising Biomarker for Predicting Recurrence in Patients with BRAF-Negative Melanoma

For patients with BRAF wild-type stage III and IV melanoma, there is an urgent clinical need to identify prognostic biomarkers and biomarkers predictive of treatment response. Circulating tumor DNA (ctDNA) is emerging as a blood-based biomarker and has shown promising results for many cancers, including melanoma. The purpose of this study was to identify targetable, tumor-derived mutations in patient blood that may lead to treatment alternatives and improved outcomes for patients with BRAF-negative melanoma. Using a CAncer Personalized Profiling by deep Sequencing (CAPP-seq) pan-cancer gene panel, ctDNA from 150 plasma samples (n=106 patients) was assessed, including serial blood collections for a subset of patients (n=16). ctDNA variants were detected in 85% of patients, all in targetable pathways, such as vascular endothelial growth factor receptor, epidermal growth factor receptor, phosphatidylinositol 3-kinase/AKT, Bcl2/mammalian target of rapamycin (mTOR), ALK/MET, and cyclin-dependent kinase 4/6. Patients with stage IV melanoma with low ctDNA concentrations, <10 ng/mL, had significantly better disease-specific survival and progression-free survival. Patients with both a high concentration of ctDNA and any detectable ctDNA variants had the worst prognosis. In addition, these results indicated that longitudinal changes in ctDNA correlated with treatment response and disease progression determined by radiology. This study confirms that ctDNA may be used as a noninvasive liquid biopsy to identify recurrent disease and detect targetable variants in patients with late-stage melanoma.

DP08 Next-generation sequencing: the future for malignant melanoma diagnostic precision? A case of a BAP1 mutation

Abstract A 48-year-old man presented to the hand surgeons with a 5-year history of a subcutaneous swelling on his right wrist, associated with paraesthesia and pain, initially thought to be a benign ganglion. Excision biopsy of the lesion on histology revealed an encapsulated pleomorphic tumour attached to a nerve, thought to represent a sarcoma. Immunohistochemistry showed a positive reaction to S100, SOX10, calretinin and synaptophysin. Ki67 showed a 20% proliferation index. Multiple pathology opinions were sought, and further testing was performed at a specialist genomic laboratory. Detailed questioning regarding family history of malignancy revealed that the patient’s mother had died from metastatic uveal melanoma, a maternal aunt had died of a brain tumour and his maternal grandmother had lung cancer. Pan-cancer gene panel DNA next-generation sequencing showed a BAP1 mutation with a very high variant allele fraction, suggestive of germline inheritance. Given the clinical history, morphology, immunoprofile and DNA result, a favoured diagnosis of metastatic malignant melanoma with a BAP1 mutation was made. The patient was referred to the specialist skin cancer multidisciplinary team meeting and reviewed by the dermatology team. Full skin check examination revealed two long-standing atypical naevi (&amp;gt; 10 years), one on the left forearm and another on his back, and these were excised urgently. Histology of the back lesion showed a polypoid melanocytic tumour comprising large epithelioid melanocytes with mild-to-moderate nuclear pleomorphism, multinucleated cells and intranuclear inclusions. This was in keeping with a pT4a nodular malignant melanoma with 7-mm Breslow thickness, associated with BAP1-inactivated syndrome. Histology of the left forearm lesion was consistent with a combined BAP1-inactivated melanocytoma, otherwise known as a ‘BAPoma’. An ophthalmology review did not identify a primary uveal melanoma. Staging scans of his chest, abdomen and pelvis and magnetic resonance imaging of his head showed no evidence of metastatic disease. The patient was referred for genetics counselling, wide local excision of the melanoma on his back, for consideration of adjuvant therapy with oncology and long-term follow-up with dermatology. BAP1 tumour predisposition syndrome is a newly recognized cancer syndrome that predisposes the patient to uveal melanoma, malignant mesothelioma, renal cell carcinoma, cutaneous melanomas and a range of other cancers (Masoomian B, Shields JA, Shields CL. Overview of BAP1 cancer predisposition syndrome and the relationship to uveal melanoma. J Curr Ophthalmol 2018; 30:102–9). BAP1-related cancers tend to occur earlier in life and are more aggressive. Our case highlights the need for early identification of germline mutations through sequencing to improve outcomes and treatment-related morbidity by early diagnosis in these rare cancer syndromes.

Perioperative circulating tumor DNA enables the identification of patients with poor prognosis in upper tract urothelial carcinoma

Perioperative systemic chemotherapy improves the prognosis of upper tract urothelial carcinoma (UTUC). The first objective of this study was to verify whether perioperative circulating tumor DNA (ctDNA) analysis using a pan‐cancer gene panel and next‐generation sequencing could identify patients with poor prognosis who require perioperative chemotherapy. Second, we investigated whether ctDNA is useful for minimal residual disease (MRD) detection and treatment monitoring in UTUC. This study included 50 patients with untreated UTUC, including 43 cases of localized UTUC. We performed targeted ultradeep sequencing of plasma cell‐free DNA (cfDNA) and buffy coat DNA and whole‐exome sequencing of cancer tissues, allowing exclusion of possible false positives. We attempted to stratify the prognosis according to the perioperative ctDNA levels in patients with localized UTUC. In patients with metastatic UTUC, ctDNA was evaluated before, during, and after systemic treatment. In total, 23 (46%) of 50 patients with untreated UTUC were ctDNA positive, and 17 (40%) of 43 patients with localized UTUC were ctDNA positive. Of the detected TP53 mutations, 19% were false positives due to clonal hematopoiesis of indeterminate potential. Among preoperative risk factors, only the preoperative ctDNA fraction>2% was a significant and independent risk factor associated with worse recurrence‐free survival (RFS). Furthermore, the existence of ctDNA early points after the operation was significantly associated with worse RFS, suggesting the presence of MRD. ctDNA also showed a potential as a real‐time marker for systemic therapy in patients with metastatic UTUC. Detection of ctDNA may indicate potential metastasis and guide decisions on perioperative chemotherapy.

Abstract PD15-01: Impact of ESR1 mutations on endocrine therapy (ET) plus alpelisib benefit in patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-), PIK3CA-mutated, advanced breast cancer (ABC) who progressed on or after prior cyclin-dependent kinase inhibitor (CDK4/6i) therapy in the BYLieve trial

Abstract PD15-01: Impact of <i>ESR1</i> mutations on endocrine therapy (ET) plus alpelisib benefit in patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-), <i>PIK3CA</i>-mutated, advanced breast cancer (ABC) who progressed on or after prior cyclin-dependent kinase inhibitor (CDK4/6i) therapy in the BYLieve trial

Abstract P5-13-03: Alpelisib + endocrine therapy (ET) in patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-), PIK3CA-mutated advanced breast cancer (ABC) previously treated with cyclin-dependent kinase 4/6 inhibitor (CDK4/6i): Biomarker analyses from the Phase II BYLieve study

Abstract Introduction: Alpelisib (ALP, a PI3Kα inhibitor and degrader) + ET has demonstrated efficacy in patients (pts) with HR+, HER2-, PIK3CA-mutated ABC progressing on/after CDK4/6i + ET in the ongoing Phase II BYLieve study (NCT03056755). The primary endpoint, in pts with centrally confirmed PIK3CA mutation in tumor tissue (modified full analysis set [mFAS]), was met in Cohorts A (ALP + fulvestrant [FUL]) and B (ALP + letrozole [LET]) with 50.4% (95% CI, 41.2%-59.6%) of 121 pts and 46.1% (36.8%-55.6%) of 115 pts alive and without disease progression at 6 mo, respectively. Activating PIK3CA mutations, occurring in ~40% of tumors, confer worse prognosis in the advanced setting. Here, we assess the correlation between progression-free survival (PFS) and other baseline biomarkers among pts in BYLieve Cohorts A and B. Methods: Enrolled pre-/postmenopausal women with HR+, HER2-, PIK3CA-mutated ABC received CDK4/6i + aromatase inhibitor (Cohort A) or FUL (Cohort B), as immediate prior treatment (Tx). Pts received ALP 300 mg PO QD + FUL 500 mg IM Q28D and C1D15 (Cohort A) or ALP 300 mg PO QD + LET 2.5 mg PO QD (Cohort B) as study Tx. This exploratory analysis of baseline biomarkers identified gene alterations using an error-connected sequencing ctDNA assay (Novartis PanCancer gene-panel) in plasma samples from pts with a centrally confirmed PIK3CA mutation in tumor tissue from Cohorts A and B. PFS was estimated using the Kaplan-Meier method in subgroups of pts based on high (≥10%) or low (&amp;lt;10%) ctDNA fraction, high (≥10) or low (&amp;lt;10) tumor mutation burden (TMB), and amplification (amp) status of chromosomes (chr) 8 or 11 amplicons (frequently observed in breast cancer and usually associated with early relapse). Results: In Cohorts A and B, 102 of 127 enrolled pts and 97 of the 126 enrolled pts were included in this analysis, respectively. In this analysis, median PFS (mPFS) was 7.3 mo in Cohort A and 5.7 mo in Cohort B, consistent with observations in the mFAS (Table 1). Pts with a low ctDNA fraction or TMB at baseline had longer mPFS than pts with high ctDNA fraction or TMB (Table 1). In addition to PIK3CA alterations observed in 73% of tumors from pts in Cohorts A and B, the most frequently altered genes in the studied population were ESR1 (27% and 28%) and TP53 (25% and 26%) in Cohorts A and B, respectively. Across both cohorts, pts whose tumors had loss of function alterations in genes known to mediate resistance to CDK4/6i (PTEN, RB1, CHD4, FAT1, NF1, ATM, CDKN2A-C) derived clinical benefit (partial response or stable disease) from ALP + ET. Amp of known amplicons on chr 8 (eg, FGFR1, TACC1) or 11 (eg, CCND1, FGF3, FGF4) were observed in 5%-8% of pts in Cohort A and 8%-11% of pts in Cohort B. Pts whose tumors had no gene amp on chr 8 and/or 11 had longer mPFS than pts whose tumors had an amp (Table 1). Similar results were observed when analyzing chr 8 (6.33 mo vs 4.92 mo) and chr 11 (6.33 mo vs 4.63 mo) separately in pts from Cohorts A plus B. Conclusion: In pts with HR+, HER2-, PIK3CA-mutated ABC, ALP was effective in the post-CDK4/6i setting regardless of ET partner and tumor genomic profile (including in presence of alterations in genes associated with CDK4/6i resistance). The absence of cross-resistance may allow pts with PIK3CA-mutated disease to benefit from ALP after disease progression on/after CDK4/6i. Table 1.mPFS in pts from BYLieve Cohorts A and B based on baseline biomarker statusCohort A (ALP + FUL)Cohort B (ALP + LET)nmPFS, mo (95% CI) nmPFS, mo (95% CI)Overall (mFASa)1,21217.3 (5.6-8.3)1155.7 (4.5-7.2)Patients with available plasma sample for the biomarker analysisb1027.37 (5.60-8.67)975.70 (3.77-7.33)ctDNA fractionHigh (≥10%)655.60 (4.00-7.37)685.43 (3.70-7.20)Low (&amp;lt;10%)37NE297.33 (5.83-11.00)TMBHigh (≥10)103.85 (0.47-8.33)154.60 (1.67-5.73)Low (&amp;lt;10)716.13 (5.43-9.60)645.63 (3.73-7.50)Chr 8 or 11 amplificationWith174.23 (1.70-6.13)213.77 (1.93-7.37)Without858.40 (5.70-9.60)766.00 (5.20-7.53)Chr, chromosome; ctDNA, circulating tumor DNA; NE, not estimable; mPFS, median progression-free survival; TMB, tumor mutation burden. aPatients who received at least 1 dose of study treatment and had centrally confirmed PIK3CA mutation by a Novartis-designated laboratory were included in the mFAS. bOnly includes patients with centrally confirmed PIK3CA mutation in tumor tissue. 1. Rugo HS, et al. Lancet Oncol. 2021;22(4):489-498; 2. Rugo HS, et al. SABCS 2020. Poster PD2-07. Citation Format: Dejan Juric, Nicholas Turner, Aleix Prat, Stephen Chia, Eva M Ciruelos, Manuel Ruiz-Borrego, Pamela Drullinsky, Florence Lerebours, Thomas Bachelot, O. Alejandro Balbin, Mukta Joshi, Estelle Roux, Christina H Arce, Murat Akdere, Hope S Rugo. Alpelisib + endocrine therapy (ET) in patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-), PIK3CA-mutated advanced breast cancer (ABC) previously treated with cyclin-dependent kinase 4/6 inhibitor (CDK4/6i): Biomarker analyses from the Phase II BYLieve study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-03.

T and NK cell abundance defines two distinct subgroups of renal cell carcinoma

ABSTRACT Renal cell carcinoma (RCC) is considered as an immunogenic cancer. Because not all patients respond to current immunotherapies, we aimed to investigate the immunological heterogeneity of RCC tumors. We analyzedthe immunophenotype of the circulating, tumor, and matching adjacent healthy kidney immune cells from 52 nephrectomy patients with multi-parameter flow cytometry. Additionally, we studied the transcriptomic and mutation profiles of 20 clear cell RCC (ccRCC) tumors with bulk RNA sequencing and a customized pan-cancer gene panel. The tumor samples clustered into two distinct subgroups defined by the abundance of intratumoral CD3+ T cells (CD3high, 25/52) and NK cells (NKhigh, 27/52). CD3high tumors had an overall higher frequency of tumor infiltrating lymphocytes and PD-1 expression on the CD8+ T cells compared to NKhigh tumors. The tumor infiltrating T and NK cells had significantly elevated expression levels of LAG-3, PD-1, and HLA-DR compared to the circulating immune cells. Transcriptomic analysis revealed increased immune signaling (IFN-γ, TNF-α via NF-κB, and T cell receptor signaling) and kidney metabolism pathways in the CD3high subgroup. Genomic analysis confirmed the typical ccRCC mutation profile including VHL, PBRM1, and SETD2 mutations, and revealed PBRM1 as a uniquely mutated gene in the CD3high subgroup. Approximately half of the RCC tumors have a high infiltration of NK cells associated with a lower number of tumor infiltrating lymphocytes, lower PD-1 expression, a distinct transcriptomic and mutation profile, providing insights to the immunological heterogeneity of RCC which may impact treatment responses to immunological therapies.

Genetic heterogeneity and predictive biomarker for pulmonary sarcomatoid carcinomas

PurposeThe aim of this study is to investigate the genetic heterogeneity (carcinomatous vs. sarcomatous components) and predictive biomarkers in patients with pulmonary sarcomatoid carcinoma (PSC). MethodsGenetic alterations and biomarkers of immunotherapy were performed in a discovery set (n = 6) of PSC. Next-generation sequencing (NGS) on a pan-cancer gene panel was applied to detect the genetic alterations in each component, and the respective mutation profiling and tumor mutation burden (TMB) were compared as well. Immunohistochemistry (IHC) assay with SP263 antibody was used to detect the protein expression of programmed death-ligand 1 (PD-L1) in each component. ResultsComparative genetic analysis revealed that the separate carcinomatous and sarcomatous components shared strikingly common mutations. TP53 (4/6, 66.7%) was the most common genetic alteration in 6 PSC patients. MET exon 14 skipping was detected in one case, accounting for 16.7%. An EZR-ROS1 fusion (EZR: intron10-ROS1: intron32) was identified in one case. The TMB of the two components was similar. Nevertheless, significantly higher PD-L1 expression was found in carcinomatous components compared to sarcomatous components. MDM2 amplification was detected in 2/6 (33.3%) of cases and STK11 mutation in 1/6 (16.7%) of cases. ConclusionsPSC containing carcinomatous and sarcomatous components had a mild heterogeneity; the two components may evolve from common ancestral cells. High PD-L1 expression suggests that immunotherapy could be used as a potential therapy for PSC patients, while patients with negative immune-responsive genes need to be screened out. Altogether, these findings further highlight that the detection of genetic alteration and PD-L1 expression plays an important role in treatment of patients with PSC.

Assessment of mutation burden after clinical intervention in pancreatic ductal adenocarcinomas (PDAC), and biliary tract cancers (BTC) via profiling circulating tumor DNA (ctDNA).

e15529 Background: PDAC and BTC have very poor prognosis with limited treatment options. Blood-based monitoring of ctDNA may have great potential to improve early detection of relapse or failure to chemotherapy and to possibly identify actionable genetic alterations. Methods: We performed targeted sequencing using NGS with Ion S5 Oncomine PanCancer gene panel to investigate mutations in 52 genes in 42 PDAC and 24 BTC patients. Blood samples were obtained from PDAC and BTC cases before the first intervention (baseline) as well as 2-4 weeks later to evaluate cfDNA mutations after surgery or chemotherapy. Actionable alterations with potential therapeutic relevance was defined as those with possible treatments within OncoKB system. We also compared the amount of cfDNA and mutant allele detection rates in pre-, intra- and post-operative samples. Results: Overall detection of ctDNA alterations in baseline samples was 74% (31/42) in PDAC cases. Alterations including SNVs and CNVs were detected among 15 genes; mutations in TP53 (53%), KRAS (41%) and SMAD4 (10%) were most commonly identified. Overall detection rate in BTC patients was 83% (20/24) comprising 9 genes; frequent mutations were observed in TP53 (58%) and KRAS (33%). Decrease in MAF (mutant allele frequency) was observed in 64% (18/28) of PDAC cases after the treatment and that of CA19-9 was observed in 50% (14/28) of the cases. In BTC pts, reduction of MAF was observed in 60% of the cases after surgery. Potentially actionable mutations were observed in 38% of PDAC samples and in 25% of BTC samples. Interestingly, the amounts of cfDNA were 4-fold higher in intraoperative samples compared to pre- and post-operative samples indicating release of cfDNA during surgery, possibly by tissue damage. Conclusions: We have shown the feasibility to assess minimum residual tumor cells or drug response by liquid biopsy in both PDAC and BTC which may improve the clinical management.

Noninvasive Detection of Microsatellite Instability and High Tumor Mutation Burden in Cancer Patients Treated with PD-1 Blockade.

Microsatellite instability (MSI) and high tumor mutation burden (TMB-High) are promising pan-tumor biomarkers used to select patients for treatment with immune checkpoint blockade; however, real-time sequencing of unresectable or metastatic solid tumors is often challenging. We report a noninvasive approach for detection of MSI and TMB-High in the circulation of patients. We developed an approach that utilized a hybrid-capture-based 98-kb pan-cancer gene panel, including targeted microsatellite regions. A multifactorial error correction method and a novel peak-finding algorithm were established to identify rare MSI frameshift alleles in cell-free DNA (cfDNA). Through analysis of cfDNA derived from a combination of healthy donors and patients with metastatic cancer, the error correction and peak-finding approaches produced a specificity of >99% (n = 163) and sensitivities of 78% (n = 23) and 67% (n = 15), respectively, for MSI and TMB-High. For patients treated with PD-1 blockade, we demonstrated that MSI and TMB-High in pretreatment plasma predicted progression-free survival (hazard ratios: 0.21 and 0.23, P = 0.001 and 0.003, respectively). In addition, we analyzed cfDNA from longitudinally collected plasma samples obtained during therapy to identify patients who achieved durable response to PD-1 blockade. These analyses demonstrate the feasibility of noninvasive pan-cancer screening and monitoring of patients who exhibit MSI or TMB-High and have a high likelihood of responding to immune checkpoint blockade.See related commentary by Wang and Ajani, p. 6887.

Detection of large rearrangements in a hereditary pan-cancer panel using next-generation sequencing

BackgroundHealthcare providers increasingly use information about pathogenic variants in cancer predisposition genes, including sequence variants and large rearrangements (LRs), in medical management decisions. While sequence variant detection is typically robust, LRs can be difficult to detect and characterize and may be underreported as a cause for hereditary cancer risk. This report describes the outcomes of hereditary cancer genetic testing using a comprehensive strategy that employs next-generation sequencing (NGS) for LR detection, coupled with LR confirmation using repeat hybrid capture NGS, microarray comparative genomic hybridization (microarray-CGH), and/or multiplex ligation-dependent probe amplification (MLPA).MethodsSequencing and LR analysis were conducted in a consecutive series of 376,159 individuals who received clinical testing with a hereditary pan-cancer gene panel from September 2013 through May 2017. NGS dosage analysis was used to evaluate potential deletions or duplications, with controls in place to exclude pseudogene reads. Samples positive for a putative LR based on NGS were confirmed using a comprehensive approach that included targeted microarray-CGH and/or MLPA analysis, with further examination as needed to ascertain the nature of the LR.ResultsA total of 3461 LRs were identified and classified as a deleterious mutation (DM), suspected deleterious mutation (SDM) or variant of uncertain significance. Pathogenic LRs (DM/SDM) accounted for the majority of LRs (67.7%), the largest proportion of which were deletions (86.1%), followed by duplications (11.3%), insertions (1.8%), triplications (0.5%), and inversions (0.3%). Several cases presented illustrate that the laboratory approach employed here can ensure consistent identification and accurate characterization of LRs. In the absence of this comprehensive testing strategy, 9% of LRs identified in this testing population might have been missed, potentially leading to inappropriate medical management in as many as 210 individuals referred for hereditary cancer testing.ConclusionsThese data show that copy number analysis using NGS coupled with confirmatory testing reliably detects and characterizes LRs. Further, LRs comprise a substantial proportion (7.2%) of pathogenic variants identified by the test. A robust and accurate LR identification strategy is an essential component of a high-quality genetic testing program, enabling clinicians to optimize patient medical management decisions.

Abstract 4252: Detecting microsatellite instability in ffpe tissue from crc subjects using next generation sequencing

Abstract Background Microsatellite instability (MSI) is a hypermutable phenotype resulting from DNA mismatch repair deficiency and is observed in up to 10-15% of early-stage colorectal cancers (CRC). It manifests as abnormal lengthening or shortening of DNA repeats at specific genomic loci. MSI-High (MSI-H) CRC patients have been shown to respond favorably towards both chemotherapy and immunotherapy and have different prognosis than MSS patients and the FDA has approved Pembrolizumab as the first cancer therapy based on a class of biomarkers rather than a cancer type. With increasing options for targeted therapy in solid tumors, being able to combine MSI testing with other cancer biomarkers for approved therapies is beneficial. We show proof of concept of a pan-cancer gene panel using an NGS-based assay and bioinformatics workflow that enables classification of MSI status along with detection of SNVs, fusions, indels and CNVs from tumor tissue samples. Unlike the gold standard PCR-based test, our method does not require a matched normal sample, thus enabling MSI detection based on tumor tissue alone. Methods Our workflow combines whole genome library preparation, hybrid capture target enrichment, high-throughput sequencing and a proprietary analysis algorithm. We expanded the number of genomic loci to improve the sensitivity of MSI detection. The analysis algorithm was trained using DNA from pure and mixed cell lines, tumor tissue and normal samples with known MSI status to classify the molecular alterations between MSI-H and MSS genotypes from sequencing reads. The algorithm leverages repeat lengths in homopolymeric MSI loci in order to predict instability of individual loci followed by aggregation using a statistical framework to make the final call on MSI status. Results The above algorithm was evaluated on a cohort of 134 stage II and stage III CRC subjects who underwent curative intent surgery. MSI status for these samples was orthogonally tested using a PCR-based MSI Analysis System (31 positives, 103 negatives) and a dMMR system. Using a pre-determined threshold, the algorithm yielded 100% sensitivity and 100% specificity on the above cohort without using matched normal tissue. A second cohort evaluated includes 47 CRC patients that are enriched for MSI positive status by virtue of their selection criteria (BRAF positive). Very high concordance on MSI status with an orthogonal method was observed. Data demonstrating high concordance with a PCR_based MSI system will be available at the presentation. Conclusions Here we present an NGS-based assay and bioinformatics workflow with robust analytical performance for MSI detection in FFPE tissue samples without a paired normal. Citation Format: Amrita Pati, Hao Wang, Hamid Mirebrahim, Seng Saelee, Joshua Lefkowitz, Sean Chien, Ashla Singh, Fergal Casey, Vera Rapoport, Xiaoju Max Ma, John Lee, Alex Lovejoy, Daniel Klass, Hans-Peter Adams. Detecting microsatellite instability in ffpe tissue from crc subjects using next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4252.

Abstract 996: In silico modeling and in vitro validation of undefined off-target of drugs in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the 5th most common and 2nd deadliest cancer worldwide. The molecular mechanism in HCC involves activated cell survival pathways, but there is a lack of the significant oncogenic drivers for targeted therapies. Thus, novel drugs and targets that can be exploited, are required. The pathways reported to be involved in hepatocarcinogenesis were retrieved from KEGG database (HCC, MAPK, calcium, p53, PI3K-Akt, Wnt, TGF-B, NAFLD and apoptosis) and a directed network with 801 nodes was created. In parallel, small molecule inhibitor drugs having at least one target protein in this network were collected from DrugBank database and integrated into the merged network. By the means of in silico perturbation attack strategies, target proteins and their interactions were calculated as the drug effectiveness, changes in the efficiency of the signaling network, in the number of feedback cycles, and in the network, functionality were identified and ranked. Finally, Brigatinib, Regorafenib, Sunitinib, Thalidomide, Pranlukast, Lenvatinib, and ChloroquineP were identified to have off-target effects specific to HCC pathways. The selected drugs along with Sorafenib were then tested and compared for their cytotoxicity on HepG2, Huh7, Mahlavu and SNU475 cells. Except Thalidomide and Pranlukast, selected drugs were significantly bioactive on the cells with IC50 values below 10µM. We then examined the expression of PanCancer gene panel (NanoString) in Mahlavu cells treated with the selected 8 drugs. As expected a number of genes and pathways were differentially altered in the presence of the drugs and multikinase inhibitors Sorafenib and Regorafenib had similar DEG patterns. In gene expression correlation analysis matrix, Sorafenib differed from the rest of the compounds particularly from Brigatinib while highest correlation was observed with Regorafenib. Although Brigatinib differs from other drugs in terms of its targets, it resulted in the highest number of differentially expressed genes. Thalidomide gene profile was very similar to Sorafenib. This data supports the possible exploitation of Thalidomide for liver cancer therapeutics. We then merged and filtered cancer-associated pathways (MAPK, STAT, PI3K, RAS, Cell Cycle, Apoptosis, Hedgehog, Wnt, DNA Damage Control, Transcriptional Regulation, Chromatin Modification, and TGF-B) based on the differentially expressed genes in Mahlavu cells treated with the 8 drugs. Previously reported genes such as APC, AXIN, CTNBB1, CAMK, TGF, NGF, ERBB2 were significantly enriched in the network along with the genes that has not been associated with liver cancer therapeutics. The enriched network analysis can be exploited for both drug repurposing and novel target identification. Citation Format: Esra Sinoplu, Nurcan Tuncbag, Deniz C. Kahraman, Rengul C. Atalay. In silico modeling and in vitro validation of undefined off-target of drugs in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 996.

Mutation concordance of matched tissue sample and blood sample: A result of 456 lung cancer patients.

e20049 Background: Cell-free DNA (cfDNA) sequencing is an important alternative for tissue sequencing when tissue samples are not available. To better study the association of a blood sample and tissue sample, we designed a study to evaluate the concordance of gene mutations of tumor tissues and matched cfDNA. Methods: From May 2017 to January 2019, 456 lung cancer patients were enrolled in this study. For each patient, a matched tumor tissue sample and blood sample were collected. DNA samples were extracted from these samples, and then sequenced with a pan-cancer gene panel. Results: The concordance ratio was computed by the number of common variants divided by the summation of common variants, the number of tissue-specific variants and number of blood-specific variants. Among these 456 patients, an overall by-variant concordance ratio of 49.7% (1457/2932) was observed. When considering only the major driver genes, the observed concordance ratios were EGFR = 45.7% (96/210), KRAS = 48.5% (147/303), ALK-fusion = 50% (8/16) and BRAF = 52.5% (21/40). The mean variant allele frequency (VAF) of common variants was significantly higher than the mean VAF of variants only detected in tissue (29.07% vs. 12.29%, p-value &lt; 2.2e-16). Conclusions: This study shows that the concordance ratio between a tissue sample and a blood sample is close to 50%. As new technologies are being developed to detect low-frequency variants better, we can expect this concordance ratio to improve in the future.

Targeted next generation sequencing identified clinically actionable mutations in patients with esophageal sarcomatoid carcinoma

BackgroundEsophageal sarcomatoid carcinoma (ESC) is a rare disease with a mixture of both carcinomatous and sarcomatous components in the tumor. Its genetic background and mechanisms of oncogenesis remain largely unknown.MethodsHere we performed targeted next generation sequencing (NGS) on a pan-cancer gene panel in 15 ESC tumors to explore their genetic alterations, and aimed to identify clinically actionable mutations for future treatment instructions.ResultsTP53 alterations were identified in all patients. Alterations in receptor tyrosine kinases (RTK) were identified in 10 out of 15 patients. Members of downstream RAS and PI3-kinase pathways are also mutated in 10 patients, and PIK3CA is the top mutated gene in these pathways. In addition, we identified mutations on histone modification genes in 5 patients, including histone acetyltransferase gene EP300 and its homologue CREBBP, lysine methyltransferase genes KMT2A and KMT2B, and lysine demethylase gene KDM5A. Finally, mismatch repair (MMR) genes and proofreading gene POLE all together were mutated in one third of the ESC patients.ConclusionsThis is the first study to unravel the mutational profile of ESC tumors. Our findings could match 9 patients to the targeted therapies currently available in clinical practice or in active clinical trials, suggesting the potential utility of targeted therapies for this rare disease in the future.